U.S. patent number 10,556,916 [Application Number 15/300,883] was granted by the patent office on 2020-02-11 for zirconium-89 oxine complex as a cell labeling agent for positron emission tomography.
This patent grant is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Services. The grantee listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Services, The United States of America, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Peter L. Choyke, Gary L. Griffiths, Noriko Sato, Haitao Wu.
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United States Patent |
10,556,916 |
Sato , et al. |
February 11, 2020 |
Zirconium-89 oxine complex as a cell labeling agent for positron
emission tomography
Abstract
The invention provides a method of preparing a .sup.89Zr-oxine
complex of the formula. The invention also provides a method of
labeling a cell with the .sup.89Zr-oxine complex and a method for
detecting a biological cell in a subject comprising administering
the .sup.89Zr-oxine complex to the subject. ##STR00001##
Inventors: |
Sato; Noriko (Potomac, MD),
Wu; Haitao (Rockville, MD), Griffiths; Gary L. (North
Potomac, MD), Choyke; Peter L. (Rockville, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Services |
Bethesda |
MD |
US |
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Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Services (Bethesda, MD)
|
Family
ID: |
52875320 |
Appl.
No.: |
15/300,883 |
Filed: |
April 1, 2015 |
PCT
Filed: |
April 01, 2015 |
PCT No.: |
PCT/US2015/023897 |
371(c)(1),(2),(4) Date: |
September 30, 2016 |
PCT
Pub. No.: |
WO2015/153772 |
PCT
Pub. Date: |
October 08, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170015685 A1 |
Jan 19, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61973706 |
Apr 1, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/16 (20130101); G01N 33/60 (20130101); C07F
7/003 (20130101); C07B 59/004 (20130101); A61K
51/1203 (20130101); A61K 51/0478 (20130101); G01N
2458/30 (20130101) |
Current International
Class: |
A61K
51/04 (20060101); A61K 51/12 (20060101); C07F
7/00 (20060101); C07B 59/00 (20060101); G01N
33/60 (20060101) |
References Cited
[Referenced By]
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WO |
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WO 2013/138696 |
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Sep 2013 |
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WO |
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WO-2013138696 |
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Sep 2013 |
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WO |
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applicant.
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Primary Examiner: Hartley; Michael G.
Assistant Examiner: Samala; Jagadishwar R
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
This invention was made with Government support under Project
Number 1ZIABC010657-11 by the National Institutes of Health,
National Cancer Institute. The Government has certain rights in
this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a U.S. national phase of International
Patent Application No. PCT/US2015/023897, filed Apr. 1, 2015, which
claims the benefit of U.S. Provisional Patent Application No.
61/973,706, filed Apr. 1, 2014, the disclosures of which are
incorporated by reference.
Claims
The invention claimed is:
1. A method of labeling a cell or microorganism with
.sup.89Zr-oxine complex comprising contacting the cell or
microorganism with a .sup.89Zr-oxine complex of the formula
##STR00003## in a buffer solution at 26.degree. C. or below,
wherein the .sup.89Zr-oxine complex permeabilizes the cell membrane
of the cell or microorganism.
2. The method of claim 1 wherein the .sup.89Zr-oxine
complex-labeled cells or microorganisms are washed free of
non-internalized .sup.89Zr.
3. The method of claim 1, wherein the cell is a healthy cell.
4. The method of claim 3, wherein the healthy cell is a T cell, a
natural killer (NK) cell, a dendritic cell, a macrophage, a
monocyte, a B cell, a myeloid cell, a platelet, a stem cell, a
progenitor cell, a mesenchymal cell, an epithelial cell, a neural
cell, a skeletal myoblast, or a pancreatic islet cell.
5. The method of claim 1, wherein the buffer solution further
comprises a second labeling agent, whereby the cell or
microorganism is labeled with both .sup.89Zr-oxine complex and the
second labeling agent.
Description
BACKGROUND OF THE INVENTION
Cell-based therapies for cancer, involving the adoptive transfer of
activated, expanded cells such as T cells, natural killer (NK)
cells and dendritic cells (DCs) have proven effective in a variety
of settings (K. Palucka et al., Nature reviews. Cancer, 2012, 12:
265-277; D. W. O'Neill et al., Blood, 2004, 104: 2235-2246; A. P.
Kater et al., Blood, 2007, 110: 2811-2828; M. Korbling et al.,
Blood, 2011, 117: 6411-6416). With the emergence of genetically
engineered T cells expressing chimeric antigen receptor and other T
cell receptors (TCR) (S. A. Grupp et al., N. Engl. J. Med. 2013,
368: 1509-1518; M. Sadelain et al., Cancer Discov., 2013, 3:
388-398; H. Shi et al., Cancer Lett., 2012, 328: 191-197), together
with interfering antibodies targeting immune-suppressive molecules,
such as PD-1, there is now great interest in cell-based therapies.
The efficacy of cell-based therapies, however, relies on the
successful migration of cells to their respective targets, tumors,
in the case of cytotoxic T cells (CTLs) or NK cells, lymphoid
organs, in the case of DC vaccines, and bone marrow (BM), in the
case of hematopoietic stem cells. Methods to monitor these
transferred therapeutic cells, however, are currently limited,
leaving uncertain the fate of these cells in patients and making it
difficult to assess the impact of cell modification on trafficking
to the target.
None of the current preclinical imaging techniques for tracking
cells are ideal for clinical use. Bioluminescence imaging (BLI)
using luciferase reporter genes and optical tagging are not
practical for whole body imaging because of the limited penetration
of light in tissue. Moreover, BLI requires gene transfection and
carries the risk of immunogenicity related to exposure to a
non-human protein. Magnetic resonance imaging with iron loaded
cells has been employed but has limited sensitivity due to negative
contrast superimposed on highly heterogeneous background.
Radiolabeling of cells has several advantages. Because the body has
no background radioactivity, very high label-to-background ratios
can be achieved and whole body monitoring is possible. Cell
labeling has classically employed .sup.111In-oxine which requires
single photon emission tomography (SPECT) imaging (M. L. Thakur et
al., J. Lab. Clin. Med., 1977, 89: 217-228; G. McAfee, M. L. et
al., J. Nucl. Med., 1976, 17: 480-487; L. Mairal et al., Eur. J.
Nucl. Med., 1995, 22: 664-670; R. J. Bennink et al., J. Nucl. Med.,
2004, 45: 1698-1704) with its inherently lower sensitivity and
resolution compared to positron emission tomography (PET) requiring
relatively high radiation doses to the labeled cells. PET is at
least ten-fold more sensitive than SPECT and therefore, has the
potential to reduce the exposure of labeled cells by at least one
log. Fluorine-18-Fluorodeoxyglucose (.sup.18F-FDG), a glucose
analog, has been used to label cells ex vivo. Because .sup.18F-FDG
labeling relies on elevated glucose metabolism, it is not suitable
for dormant or inactivated cells. Moreover, the half-life of
.sup.18F (109.7 min) significantly limits the amount of time for
cell tracking. Finally, .sup.18F-FDG is released from the cells by
phosphatase activity (C. Botti et al., Eur. J. Nucl. Med., 1997,
24: 497-504; E. Wolfs et al., J. Nucl. Med., 2013, 54: 447-454)
leading to non-specific signals. In order to track cells for at
least several days, a positron emitting radioisotope with a longer
half-life is required.
Thus, there remains a need in the art for methods for labeling
cells with an agent that allows for tracking the cells for at least
several days without significantly interfering with cell survival,
proliferation, or function.
BRIEF SUMMARY OF THE INVENTION
The invention provides a method of preparing a .sup.89Zr-oxine
complex of the formula:
##STR00002## comprising (i) combining a solution of oxine and
.sup.89ZrCl.sub.4 in hydrochloric acid at room temperature (e.g.,
16-26.degree. C., 20.degree. C..+-.2.degree. C., around 20.degree.
C.) to form a mixture, (ii) adding an alkaline solution to the
mixture in an amount effective to neutralize the mixture, (iii)
generating .sup.89Zr-oxine complex, and optionally, (iv) extracting
.sup.89Zr-oxine into an organic solvent to isolate the
.sup.89Zr-oxine complex.
The invention also provides a method of labeling a cell with
.sup.89Zr-oxine comprising contacting the cell with the
.sup.89Zr-oxine complex in a buffer solution at room temperature or
below.
The invention further provides a kit for labeling biological cells
for PET-imaging, comprising (a) a first component comprising
.sup.89Zr, (b) a second component comprising oxine, (c) a third
component comprising an alkaline solution, and (d) instructions for
use.
The invention additionally provides a method of detecting a
biological cell or a microorganism in a subject comprising
administering to the subject a labeled biological cell or
microorganism comprising the .sup.89Zr-oxine complex and examining
at least a portion of the subject by PET imaging, thereby detecting
the labeled biological cell or microorganism in the subject.
The invention also provides a method of transplanting a biological
cell into a subject comprising (a) administering to the subject a
labeled biological cell comprising the .sup.89Zr-oxine complex, (b)
examining at least a portion of the subject by PET imaging, (c)
detecting the migration pattern and/or cellular distribution
pattern of the labeled biological cell in the subject, and (d)
optionally administering additional biological cells.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIG. 1A illustrates the cell labeling efficiency of .sup.89Zr-oxine
complex in EL4 cells in PBS, serum free medium or in complete
medium.
FIG. 1B illustrates the cell labeling efficiency of .sup.89Zr-oxine
complex in dendritic cells (DCs), naive cytotoxic T cells (CTLs),
activated CTLs, natural killer (NK) cells, bone marrow (BM) cells,
and EL4 murine lymphoma cells.
FIG. 1C illustrates the specific activity of DCs, naive CTLs,
activated CTLs, NK cells, BM cells, and EL4 murine lymphoma cells
labeled with .sup.89Zr-oxine complex.
FIG. 2A illustrates viability of DCs with and without
.sup.89Zr-oxine complex labeling up to 5 days after the labeling in
culture with granulocyte macrophage colony stimulation factor
(GM-CSF).
FIG. 2B shows that CTLs with and without .sup.89Zr-oxine complex
labeling underwent similar proliferation upon TCR stimulation
followed by a contraction phase after withdrawal of the TCR
stimulation.
FIG. 2C shows that .sup.89Zr-oxine complex associated with DCs
paralleled the number of surviving DCs.
FIG. 2D shows that .sup.89Zr-oxine complex was retained in the CTLs
during the rapid proliferation, but decreased during the
contraction phase in which cell number decreased.
FIGS. 3A-3E shows that .sup.89Zr-oxine complex-labeled DCs
upregulated CD80 (FIG. 3A), CD86 (FIG. 3B), and CD40 (FIG. 3C), as
well as MHC molecules of class I (FIG. 3D) and class II (FIG. 3E)
similarly to non-labeled control DCs when DCs were activated by a
Toll-like receptor ligands, lipopolysaccharide (LPS).
FIG. 3F shows that .sup.89Zr-oxine complex-labeled DCs were capable
of presenting the antigen and inducing T cell activation in vivo as
well as non-labeled DCs.
FIGS. 4A-4C shows that .sup.89Zr-oxine complex-labeled CTLs were
activated upon TCR stimulation as well as non-labeled cells as
indicated by induction of CD44 (FIG. 4A), CD69 (FIG. 4B), and CD25
expression (FIG. 4C).
FIGS. 4D-4E shows that the .sup.89Zr-oxine complex-labeled CTLs
were capable of producing IFN-.gamma. (FIG. 4D) and IL-2 (FIG. 4E)
upon TCR activation.
FIG. 5A shows that .sup.89Zr-oxine complex-labeled DCs injected
into mice via the tail vein initially distributed in the lungs and
gradually migrated to the spleen and liver by day 1, detected by
microPET imaging.
FIG. 5B shows that CTLs labeled with .sup.89Zr-oxine complex mainly
distributed in the spleen after migrating out from the lungs after
injection into mice via the tail vein, detected by microPET
imaging.
FIG. 6A depicts microPET imaging of .sup.89Zr-oxine complex labeled
OT-1 TCR transgenic CTLs targeting melanoma tumors expressing the
nominal antigen inoculated in the flank.
FIG. 6B shows that the .sup.89Zr-oxine complex labeled OT-1 CTLs
(.box-solid.) retained cytotoxic function and induced regression of
melanoma tumors expressing the nominal antigen after the transfer
compared to untreated subjects (.largecircle.).
FIGS. 7A and 7B illustrate the biodistribution of .sup.89Zr-oxine
complex-labeled DCs at 1 and 7 days, respectively, after the
transfer to WT mice.
FIG. 8A demonstrates that BM cells labeled with .sup.89Zr-oxine and
cultured in stem cell factor (SCF), Fms-related tyrosine kinase 3
ligand (FLT3L), and thrombopoietin (TPO) survived and
proliferated.
FIG. 8B depicts that total .sup.89Zr activity associated with cells
decreased as the cells died during the 0-2 day period, but remained
stable as the rate of cell death decreased and cells began to
proliferate after day 2.
FIG. 8C demonstrates that specific activity of the .sup.89Zr-oxine
labeled BM cells decreased in the cells labeled with the lower dose
as cells proliferated, but remained about the same in cells labeled
with the higher dose.
FIG. 9A demonstrates that labeling did not alter the expression of
sca-1, CD117, and lineage markers in BM cells.
FIGS. 9B and 9C demonstrate that .sup.89Zr-oxine labeled BM cells
and non-labeled cells cultured with GM-CSF (FIG. 9B) or IL-15 (FIG.
9C) differentiated into mature DCs (FIG. 9B) and NK/NK-T cells
(FIG. 9C), respectively, in a comparable manner.
FIG. 10A is a serial microPET/CT imaging of .sup.89Zr-oxine labeled
BM cells that revealed a rapid trafficking of BM cells through the
lungs to the BM, spleen, and liver.
FIG. 10B illustrates the kinetics of BM cell migration to the BM in
the spine (.circle-solid.), lungs (.box-solid.), liver
(.tangle-solidup.), and spleen (.circle-solid.) analyzed from the
PET/CT images of non-irradiated and irradiated recipient mice
(n=3).
FIG. 11A illustrates that .sup.89Zr-oxine microPET/CT imaging
demonstrated CXCR4 dependent BM homing and retention within the BM
of transferred BM cells. Plerixafor or plerixafor/G-CSF were
injected 15 min before and 1 day after the cell transfer. "Pre"
indicates 1 hour prior to plerixafor/G-CSF injection and "Post" is
1 hour after plerixafor/G-CSF injection.
FIG. 11B is a kinetics analysis of the cell migration that
demonstrated inhibition of BM homing with plerixafor at the 0-2 h
time point and prolonged inhibition with plerixafor/G-CSF. All
recipient mice received a lethal whole-body irradiation at 9.5 Gy
24 h prior to cell transfer. "Pre" indicates 1 hour prior to
plerixafor/G-CSF injection and "Post" is 1 hour after
plerixafor/G-CSF injection. Asterisks indicate a statistical
significance between control 0 h group: *P<05; **P<0.01;
***P<0.001; <****P<0.0001.
FIG. 12A demonstrates that donor GFP.sup.+ BM cells labeled with
.sup.89Zr-oxine were mobilized into the blood 1 day following
plerixafor/G-CSF treatment. Asterisks indicate statistical
significance. **P<0.01.
FIG. 12B is a flow cytometry analysis that confirmed mobilized
donor GFP.sup.+ BM cells in the circulation with some variations
among the mice. Control (i) and mobilized (ii-iii) mice (n=4).
FIG. 13A demonstrates that donor derived cells reconstituted the BM
of the hosts received BM ablation prior to the transplantation, but
not in the non-BM ablated hosts.
FIG. 13B is a flow cytometry analysis of splenocytes harvested from
recipient mice 10 weeks following cell transfer demonstrating that
BM cells had differentiated into DCs (CD11c.sup.+), NK cells
(CD3.sup.-NK1.1.sup.+), and T cells (CD3.sup.+NK1.1.sup.-) in BM
ablated but not in non-ablated recipient mice.
DETAILED DESCRIPTION OF THE INVENTION
The .sup.89Zr-oxine complex described herein comprises .sup.89Zr
and oxine. Oxine is 8-hydroxyquinoline. .sup.89Zr is produced, for
example via an .sup.89Y(pn).sup.89Zr or .sup.89Y(d,2n).sup.89Zr
reaction. In an .sup.89Y(pn).sup.89Zr reaction, a proton beam with
about 14 MeV energy is used to bombard a target such as a yttrium
foil mounted onto an aluminum/copper disk. In a
.sup.89Y(d,2n).sup.89Zr reaction, about a 14 MeV deuteron beam is
used to irradiate a yttrium pellet. The .sup.89Zr is liberated from
the target, typically in an acidic solution such as an oxalic acid
solution. Thus, in an embodiment, the .sup.89Zr is provided as an
.sup.89Zr-oxalic acid solution. In an embodiment, the
.sup.89Zr-oxalic acid solution can be loaded onto a chromatography
column, such as a C-18 SEP-PAK.TM. cartridge (Waters, Milford,
Mass.), and then eluted with aqueous hydrochloric acid to provide
.sup.89Zr as .sup.89ZrCl.sub.4.
.sup.89Zr-oxine complex can be generated in aqueous solution by
conjugating oxine to .sup.89Zr. In an embodiment, a solution of
oxine in, e.g., dilute hydrochloric acid, can be combined with a
solution of .sup.89ZrCl.sub.4 optionally in the presence of a
surfactant, such as polysorbate 80 ("TWEEN.TM. 80") or functional
equivalents thereof. The aqueous solution of .sup.89Zr-oxine
complex is suitable for use in the inventive methods disclosed
herein. Optionally, the .sup.89Zr-oxine complex can be extracted
into an organic solvent such as chloroform to isolate the
.sup.89Zr-oxine complex.
In an embodiment, the .sup.89Zr-oxine complex is used to label
biological cells, particularly mammalian cells. Also included
herein is a biological cell labeled with .sup.89Zr-oxine complex.
The term "biological cell" as used herein, refers to a cellular
structure having biological functionality including, but not
limited to, production of biological proteins, and/or induction of
extracellular ligand binding sites. A biological cell can be
naturally occurring or modified and is preferably viable. In a
preferred embodiment, the biological cell is a healthy cell. In
certain preferred embodiments, the biological cell is a T cell, a
natural killer (NK) cell, a dendritic cell, a macrophage, a
monocyte, a B cell, a myeloid cell, a platelet, a stem cell, a
progenitor cell, a mesenchymal cell, an epithelial cell, a neural
cell, a skeletal myoblast, or a pancreatic islet cell. Non-limiting
examples of suitable stem cells include bone marrow-derived stem
cells such as hemopoeitic stem cells, embryonic stem cells, adult
stem cells, mesenchymal stem cells, epidermal stem cells,
endothelial stem cells, endothelial progenitor cells, resident
cardiac stem cells, induced pluripotent stem cells, adipose-derived
stem cells, amniotic fluid stem cells, uterine stem cells, neural
stem cells, neural progenitor cells, cancer stem cells (e.g., a
leukemic hematopoietic stem cells, solid tumor stem cells),
umbilical cord blood stem cells, or any combination thereof. In an
embodiment, the biological cell is a bone marrow cell (e.g., a stem
cell, progenitor cell). Further, a biological cell can be a
cancerous cell, for example, a breast cancer cell or a leukemic
cell. In an embodiment, the biological cell is genetically
engineered in order to stay immortalized, to enhance the targeting
property, or to enhance cellular function, or is a genetically
altered vector-producing cell.
In an embodiment, the biological cell is in cell culture, that is,
the cells are ex vivo. When in cell culture, the biological cell is
typically in a vessel such as a culture dish that contains a
nutrient broth called a culture medium. In an embodiment,
biological cells are labeled by contacting the biological cells
with a solution/suspension containing the .sup.89Zr-oxine complex
in an amount and for a time sufficient to label the cells with the
.sup.89Zr-oxine complex. Culture medium is defined as a liquid that
covers biological cells in a culture dish and that contains
nutrients to nourish and support the cells. Culture medium may
include growth factors and other additives to produce desired
changes in the cells.
In another embodiment, a microorganism can be tracked by labeling
the microorganism with the .sup.89Zr-oxine complex and subsequently
imaged (e.g., imaged in a subject). The microorganism can be any
suitable single celled or multicellular microorganism, such as an
infectious pathogen or human microbiome. Examples of suitable
microorganisms include viruses, viroids, bacteria (e.g., commensal
bacteria), parasites, archaea, protozoa, fungus, algae, yeasts, and
rotifers. In a particular embodiment, the microorganism is
commensal bacteria (e.g., normal microflora, indigenous
microbiota).
As used herein labeling of a cell with .sup.89Zr-oxine complex
means that the complex is inside the cell or microorganism or
associated with the cell or microorganism such that the complex,
and thus the cell or microorganism, can be detected such as by PET
imaging.
In an embodiment, after labeling with the .sup.89Zr-oxine complex,
the cells or microorganisms are washed with a solution containing a
chelator such as deferoxamine mesylate to remove any free
.sup.89Zr. Additional chelators include Deferasirox, an iron
chelating medication that comes in a tablet form, hydroxyethyl
starch deferoxamine (HESdeferoxamine), EDTA, and DTPA.
It was determined experimentally that cells and microorganisms can
be effectively labeled with the .sup.89Zr-oxine complex did not
significantly alter cellular phenotypes, survival, proliferation,
and/or function. Such improvements enable the tacking of labeled
cells and microorganisms for a desired time period (e.g., several
days). In an embodiment, the .sup.89Zr-oxine complex-labeled cells
or microorganisms are administered to a subject, and the labeled
cells or microorganisms are then imaged by PET. For use in a
therapeutic regimen, methods of administration/delivery of cells or
microorganisms include injections and use of special devices to
implant cells or microorganisms in various organs. The present
disclosure is not limited to any particular delivery method. For
example, labeled cells or microorganisms can be imaged following
either a focal implantation directly into tissues, subcutaneously,
subdermally, or by intravenous injection. Exemplary injection
techniques include intravenous, intra-arterial, intraperitoneal
and/or direct tissue injection including dermal and subdermal.
Cells or microorganisms can be inserted into a delivery device that
facilitates introduction by injection or implantation into the
subjects. Such delivery devices include tubes, e.g., catheters, for
injecting cells or microorganisms and fluids into the body of a
recipient subject. In an embodiment, the tubes additionally have a
needle, e.g., a syringe, through which the cells or microorganisms
can be introduced into the subject at a desired location. The cells
or microorganisms can be prepared for delivery in a variety of
different forms. For example, the cells or microorganisms can be
suspended in a solution or gel or embedded in a support matrix when
contained in such a delivery device. Cells or microorganisms can be
mixed with a pharmaceutically and/or diagnostically acceptable
carrier or diluent in which the cells or microorganisms remain
viable. Pharmaceutically and/or diagnostically acceptable carriers
and diluents include saline, aqueous buffer solution, solvents,
and/or dispersion media. The use of such carriers and diluents is
well known in the art. The solution is preferably sterile and
fluid. In specific embodiments, the solution is stable under the
conditions of manufacture and storage and preserved against the
contaminating action of microorganisms such as bacteria and fungi
by use of preservatives. Solutions can be prepared by incorporating
cells or microorganisms as described herein in a pharmaceutically
and/or diagnostically acceptable carrier or diluent and, as
required, other ingredients enumerated above, followed by filtered
sterilization.
In certain embodiments, the .sup.89Zr-oxine complex-labeled cells
or microorganisms further comprise an MR imaging agent such as a
superparamagnetic agent or fluorine-19 (.sup.19F) agent to allow
for PET-MRI. An exemplary superparamagnetic agent is a
superparamagnetic nanoparticle optionally associated with a
polymer. Superparamagnetism means a form of magnetism, which
appears in small ferromagnetic or ferromagnetic nanoparticles. Like
the paramagnetic materials, the superparamagnetic materials do not
maintain their magnetism in the absence of an externally applied
magnetic field. Superparamagnetic nanoparticles are particles
having at least one dimension of 1 nm to 100 nm (e.g., at least 1
nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm,
at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at
least 90 nm, and/or less than 100 nm, less than 90 nm, less than 80
nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40
nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 5
nm, or any combination thereof) and that exhibit superparamagnetic
properties. Superparamagnetic nanoparticles include iron oxide,
dysprosium oxide, gadolinium oxide, manganese oxide, gold oxide,
silver oxide and combinations thereof. Iron oxides include, for
example Fe.sub.3O.sub.4, .gamma.-Fe.sub.2O.sub.3, FeOOH, and
.alpha.-Fe.sub.2O.sub.3. The nanoparticle can be any shape,
including sphere, rod, or platelet. An exemplary superparamagnetic
agent is FERAHEME.TM.. .sup.19F can be used as anionic or cationic
emulsions to label cells.
In an embodiment, the superparamagnetic nanoparticle is associated
with a polymer. In an embodiment, the polymer substantially coats
at least a portion of the nanoparticle. Without wishing to be held
to any particular theory or mechanism, it is believed that the
polymer can facilitate in vivo transport of the nanoparticle
throughout a subject, and facilitate uptake and retention of the
nanoparticles by tissues and cells. The polymer can be a natural or
a synthetic polymer. Exemplary synthetic polymers include
poly(acrylic acid), poly(methacrylic acid), poly(ethylmethacrylic
acid), poly(butylmethacrylic acid), poly(laurylmethacrylic acid),
poly(hydroxyethylmethacrylic acid), poly(hydroxypropylmethacrylic
acid), poly(acrylamide), poly(isocyanate), poly(styrene),
poly(ethyleneimine), poly(siloxane), poly(glutamic acid),
poly(aspartic acid), poly(lysine), polypropylene glycol, poly(vinyl
alcohol), poly(vinyl pyrrolidone), polyethylene oxide, derivatives
thereof, and combinations thereof. Exemplary natural or
semi-synthetic polymers include chitosan, dextran, carboxymethyl
dextran, cellulose, hyaluronic acid, alginate, their carboxymethyl
or other derivatives, and combinations thereof. The polymers can be
modified to include functional groups such as carboxymethyl or
hydroxymethyl groups. The polymers can also be crosslinked or
grafted to other polymers. In an embodiment, the polymer is a
dextran such as a carboxymethylated dextran. In a specific
embodiment, the polymer associated with the superparamagnetic
nanoparticle is polyglucose sorbitol carboxymethyl ether.
In a specific embodiment, the superparamagnetic nanoparticle is
ferumoxytol (FERAHEME.TM.), a superparamagnetic magnetite
(Fe.sub.3O.sub.4) nanoparticle associated with a low molecular
weight semi-synthetic carbohydrate, polyglucose sorbitol
carboxymethyl ether, with potential anti-anemic and imaging
properties. Ferumoxytol is commercially available as an aqueous
colloidal drug. The overall colloidal particle size in the product
commercially available from AMAG Pharmaceuticals is 17-31 nm in
diameter. The chemical formula of FERAHEME.TM. is
Fe.sub.5874O.sub.8752C.sub.11719H.sub.18682O.sub.9933Na.sub.414
with an apparent molecular weight of 750 kDa.
In another embodiment, the superparamagnetic nanoparticle is
ferumoxide (FERIDEX IV.TM.), a non-stoichiometric magnetite
associated with dextran.
In an embodiment, for use as MRI contrast agents, it is preferred
that the superparamagnetic complexes have a neutral or positive
zeta potential in water and a negative zeta potential in balanced
isotonic salt solutions. In an embodiment, the superparamagnetic
complexes, such as heparin-protamine-ferumoxytol ("HPF") complexes,
have a positive zeta potential of 5 to 25, more specifically 10 to
20 mV in water and/or 0 to -15 mV in isotonic salt solutions.
In certain embodiments, the .sup.89Zr-oxine complex-labeled cells
or microorganisms further comprise an MR imaging agent comprising
fluorine-19 (.sup.19F). Since biological tissues have negligible
endogenous fluorine content, in vivo .sup.19F MRI can provide an
effective means of detecting labeled cells or microorganisms.
In certain embodiments, the cells or microorganisms can be
contacted ex vivo with a fluorocarbon imaging reagent under
conditions such that the fluorocarbon imaging reagent becomes
associated with the cell or microorganism. In certain embodiments,
the .sup.19F labeling agents can be linear or cyclic
perfluoropolyethers or linear or cyclic perfluorohydrocarbons.
Examples of suitable .sup.19F labeling agents include, but are not
limited to, perfluorocarbon-based emulsion, perfluoropolyether
emulsion, perfluoro-15-crown-5 ether (PRCE), perfluorooctyl bromide
(PFOB), perfluorodecalin (PFD), trans-bis-perfluorobutyl ethylene
(F-33E). These agents can label cells or microorganisms without
using additional reagents to facilitate the intake.
In certain embodiments, the cells or microorganisms can be
contacted with the .sup.19F labeling agent in the presence of a
reagent that enhances uptake of the .sup.19F labeling agent. The
.sup.19F labeling agent can be provided in the form of an emulsion
in water containing a surfactant. Suitable surfactants can be
nonionic, anionic, or cationic surfactants, and also include
phospholipids. Examples of suitable nonionic surfactants include
ethylene oxide-propylene oxide block copolymers. Examples of
suitable cationic surfactants include cationic lipids and protamine
sulfate. An example of a suitable phospholipid is egg lecithin.
Emulsions can be prepared as described in Jacoby et al., NMR
Biomed., 2014, 27: 261-271, and other examples of suitable .sup.19F
labeling agents and techniques are disclosed in Srinivas et al.,
Trends Biotechnol. 2010 Jul. 28(7): 363-370 and Temme et al.,
Journal of Leukocyte Biology Volume 95, April 2014, 689-697, the
disclosures of which are incorporated totally herein by
reference.
The term "detect" includes imaging to ascertain the presence or
absence of a labeled molecule, cell, or microorganism, particularly
by a PET technique. In an embodiment, PET, PET/CT, and/or PET-MRI
allows the determination of the extent of migration of the
.sup.89Zr-oxine complex-labeled cells or microorganisms, and/or
whether more cells are needed for repair or replacement of damaged
tissue. The imaging information obtained will also allow clinicians
to associate the clinical findings and therapeutic index as it
relates to the presence of the cells or the mechanisms behind their
workings in order to optimize the therapeutic regimen or the
tracking of cells or microorganisms.
The .sup.89Zr-oxine complex-labeled cells disclosed herein will
allow the direct transplantation of the .sup.89Zr-oxine
complex-labeled cells, non-limiting examples of which include T
cell, natural killer (NK) cell, dendritic cell, macrophage,
monocyte, B cell, myeloid cell, platelet, stem cell, progenitor
cell, mesenchymal cell, epithelial cell, neural cell, skeletal
myoblast, or pancreatic islet cell, into tissues for purposes of
immunotherapy, the treatment of malignancies and/or viral
infections, investigational research purposes, monitor the healing
of injuries, and/or to track the migration pattern and/or cellular
distribution of the labeled cells noninvasively and repeatedly as
necessary. In an embodiment, immune cells, not limited to,
dendritic cells, T cells, NK cells, or other genetically altered
cells are labeled with the .sup.89Zr-oxine complex disclosed herein
to non-invasively monitor their trafficking into tissues or lesions
in autoimmune or inflammatory diseases, ischemic diseases of the
heart and central nervous system, genetically deficient disease
states, and into malignancy as part of a therapeutic approach.
Local administration of the .sup.89Zr-oxine complex can be used in
conjunction with systemic immunotherapy, that is, immunotherapy or
vaccine therapy provided to the whole body.
As described herein, when .sup.89Zr-oxine complex-labeled cells or
microorganisms also comprise an MR imaging agent, nuclear magnetic
resonance techniques can be used to detect populations of MR
imaging agent-labeled cells or microorganisms. MRI can include more
sophisticated measurements, including quantitative measurements and
two- or three-dimensional image generation. For example, MRI can be
used to generate images of such cells. In many instances, the
labeled cells are administered to a living subject. Following
administration of the cells or microorganisms, some portion of the
subject, or the entire subject, is examined by MRI to generate an
MRI data set. A "data set" means raw data gathered during magnetic
resonance probing of the subject material, as well as information
processed, transformed, or extracted from the raw data. Examples of
processed information include two-dimensional or three-dimensional
pictorial representations of the subject material.
MRI examination can be conducted according to a suitable
methodology known in the art. Many different types of MRI pulse
sequences, or the set of instructions used by the MRI apparatus to
orchestrate data collection, and signal processing techniques
(e.g., Fourier transform and projection reconstruction) have been
developed over the years for collecting and processing image data.
The reagents and methods described herein are not tied to any
particular imaging pulse sequence or processing method of the raw
NMR signals. For example, MRI methods include spin-echo,
stimulated-echo, gradient-echo, free-induction decay based imaging,
and any combination thereof. The development of new and improved
pulse sequence and signal processing methods is a continuously
evolving field, and persons skilled in the art can devise multiple
ways to image the labeled cells in their anatomical context.
In an embodiment, the .sup.89Zr-oxine complex-labeled cells or
microorganisms can also comprise an optical dye, such as a
fluorescent dye containing a fluorophore, to allow for detecting
the cells or microorganisms using optical techniques. Suitable
optical techniques include, but are not limited to, optical imaging
such as fluorescence imaging including near-infrared fluorescence
(NIRF) imaging, bioluminescence imaging, or combinations thereof. A
typical fluorophore is, for example, a fluorescent aromatic or
heteroaromatic compound, such as a pyrene, an anthracene, a
naphthalene, an acridine, a stilbene, an indole or benzindole, an
oxazole or benzoxazole, a thiazole or benzothiazole, a
4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a
carbocyanine, a carbostyryl, a porphyrin, a salicylate, an
anthranilate, an azulene, a perylene, a pyridine, a quinoline, a
coumarin (including hydroxycoumarins and aminocoumarins and
fluorinated derivatives thereof). Near infrared-emitting probes
exhibit decreased tissue attenuation and autofluorescence (Burns et
al., Nano Letters, 2009, 9 (1), 442-448).
Non-limiting fluorescent compounds that can be used in the present
invention include, Cy5, Cy5.5 (also known as Cy5++), Cy2,
fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3,
Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640,
LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine,
rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine
6G (R6G), the rhodamine derivative JA133, Alexa Fluorescent Dyes
(such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa
Fluor 555, and Alexa Fluor 647), 4',6-diamidino-2-phenylindole
(DAPI), Propidium iodide, AMCA, Spectrum Green, Spectrum Orange,
Spectrum Aqua, Lissamine, and fluorescent transition metal
complexes, such as europium. Fluorescent compound that can be used
also include fluorescent proteins, such as GFP (green fluorescent
protein), enhanced GFP (EGFP), blue fluorescent protein and
derivatives (BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent
protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow
fluorescent protein and derivatives (YFP, Citrine, Venus, YPet),
and dyes disclosed in WO 2008/142571, WO 2009/056282, and WO
99/22026, each of which is incorporated herein in its entirety. In
another embodiment, the optical dye is conjugated to a
nanoparticle, for example, a silica-based nanoparticle.
Also provided are compositions comprising an .sup.89Zr-oxine
complex, including cells or microorganisms, such as an
.sup.89Zr-oxine complex-labeled cell or microorganism. The
compositions can be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration can be topical, pulmonary,
intratracheal, intranasal, epidermal, and transdermal, intradermal,
oral, or parenteral. Parenteral administration includes
intravenous, intraarterial, subcutaneous, intraperitoneal,
intramuscular injection or infusion, or intracranial, e.g.,
intrathecal or intraventricular administration.
Compositions and formulations for parenteral, intrathecal, or
intraventricular administration may include sterile aqueous
solutions that may also contain buffers, diluents, and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds, and other pharmaceutically and/or
diagnostically acceptable carriers or excipients.
Compositions include, but are not limited to, solutions, emulsions,
and liposome-containing formulations. These compositions can be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids, and
self-emulsifying semisolids.
The formulations, which may conveniently be presented in unit
dosage forms, can be prepared according to conventional techniques
well known in the art. Such techniques include the step of bringing
into association the components with the pharmaceutical carrier(s)
or excipient(s). In general, the formulations are prepared by
uniformly and intimately bringing into association the active
ingredients with liquid carriers or finely divided solid carriers
or both, and then, if necessary, shaping the product.
The compositions may additionally contain other adjunct components
conventionally found in pharmaceutical compositions. Thus, for
example, the compositions may contain additional, compatible,
pharmaceutically-active materials such as, e.g., antipruritics,
astringents, local anesthetics, or anti-inflammatory agents, or may
contain additional materials useful in physically formulating
various dosage forms of the compositions of the present invention,
such as dyes, flavoring agents, preservatives, antioxidants,
opacifiers, thickening agents, and stabilizers. However, such
materials, when added, should not unduly interfere with the
biological activities of the components of the compositions of the
present invention. The formulations can be sterilized and, if
desired, mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizer, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, colorings, flavorings and/or
aromatic substances and the like which do not deleteriously
interact with the active components of the formulation.
PET imaging has clear advantages for labeling cells for cell-based
therapies. Compared to optical and magnetic resonance imaging using
superparamagnetic agent, there is no background signal in PET.
Compared to SPECT imaging, PET has a ten fold better sensitivity
resulting in lower doses of ionizing radiation to these sensitive
cells. An added benefit of PET is improved resolution compared to
SPECT. Moreover, in the clinical setting, PET/CT can be performed
in a shorter time period than SPECT/CT. The present invention
provides advantageously a long lived PET emitter, with .sup.89Zr
having a half-life of 3.27 days, that is capable of safely labeling
hematopoietic cells.
Herein it has been demonstrated that .sup.89Zr-oxine complex, when
incubated with at least four types of hematopoietic cells, can
label the cells with sufficient efficiency to enable imaging. Since
the labeling occurred at 4.degree. C., it can be inferred that
.sup.89Zr-oxine complex does not require active cell transport and
indicates that oxine conjugates can permeabilize the cell membrane.
Moreover, optimal labeling occurred at room temperature (e.g.,
16-26.degree. C., 20.degree. C..+-.2.degree. C., around 20.degree.
C.), suggesting that this method of labeling should be readily
translatable to the clinic. Once labeled the cells demonstrated
equal viability and proliferation compared to unlabeled cells. In
addition, advantageously cell expression assays performed before
and after .sup.89Zr-oxine complex labeling indicated that
functionality of cells was maintained after labeling.
Imaging and biodistribution of .sup.89Zr-oxine complex-labeled
cells demonstrated preferential uptake in organs according to the
cell type. For instance, after clearing the lungs, DC distributed
in the liver and spleen, whereas CTLs distributed exclusively to
the spleen and lymph nodes on microPET imaging. Additionally,
activated OT-1 CD8 cells targeted ovalbumin expressing tumors and
accumulated within the tumor on microPET ultimately resulting in a
dramatic decrease in tumor size. These images could be obtained at
remarkably low dose levels (e.g. 145-185 kBq or 4-5 .mu.Ci). Thus,
.sup.89Zr-oxine complex labeling resulted in the ability to track a
variety of cell types using microPET scans.
The ability to label and track a wide variety of cells is of
potential importance to improving cell based therapies. For
instance, in the case of bone marrow transplants, modifying
conditions to result in increased uptake of transplanted cells in
the bone marrow is considered desirable. .sup.89Zr-oxine complex
could be used to determine whether modifications to cells, methods
of delivery (e.g. intravenous vs. intrabone marrow), and adjuvant
therapies, increases the number of engrafted cells. For tumor
targeting therapies, the effect of cell modification on tumor
targeting has clear implications for the success of these
therapies. Thus, the inventive method could be a useful tool across
a broad range of cell-based therapies.
In an embodiment, the invention provides a kit for the labeling of
biological cells or microorganisms. The kit comprises (a) a first
component comprising .sup.89Zr, (b) a second component comprising
oxine, (c) an alkaline solution, and (d) instructions for use. In
an embodiment, the kit further comprises at least one other
labeling agent (e.g., a superparamagnetic nanoparticle, .sup.19F
and/or an optical dye). In an embodiment, the .sup.89Zr is in a
first container and the oxine is in a second container. In an
embodiment, the alkaline solution is in a third container. In any
of these embodiments, the kit can further comprise at least one
surfactant, such surfactants known in the art and those described
herein.
For purposes of the present invention, the term "subject"
preferably is directed to a mammal. Mammals include, but are not
limited to, the order Rodentia, such as mice, and the order
Logomorpha, such as rabbits. It is preferred that the mammals are
from the order Carnivora, including Felines (cats) and Canines
(dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the
order Perssodactyla, including Equines (horses). It is most
preferred that the mammals are of the order Primates, Ceboids, or
Simioids (monkeys) or of the order Anthropoids (humans and apes).
An especially preferred mammal is the human.
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
Materials and Methods
Mice and Cells
C57BL/6 (WT), Rag1 deficient (Rag1KO) and OT-1 T cell receptor
transgenic mice against ovalbumin (OVA) were purchased from Jackson
Laboratories (Bar Harbor, Me.). Male and female C57BL/6 wild type
(expressing CD45.2) and congenic (expressing CD45.1) mice and green
fluorescence protein (GFP) transgenic mice were purchased from
Jackson Laboratories (Bar Harbor, Me.). All animal experiments were
performed in compliance with the NIH Guide for the Care and Use of
Laboratory Animals.
Cells were grown in RPMI 1640 medium (Life Technologies, Grand
Island, N.Y.) supplemented with 10% fetal calf serum (FCS), 1%
penicillin-streptomycin and 50 .mu.M 2-mercaptoethanol
(Sigma-Aldrich, St. Louis, Mo.). DCs were differentiated from the
bone-marrow of WT mice using granulocyte colony stimulating factor
(20 ng/ml, GM-CSF, Peprotech, Rocky Hill, N.J.). Activation of DCs
was induced by lipopolysaccharide (LPS, 5 ng/ml, Sigma-Aldrich)
overnight on day 5 or 6, with or without OVA peptide (SIINFEKL, 1
.mu.g/ml, AnaSpec, Fremont, Calif.), and used on the following day.
Naive CD8 T cells were purified from the spleen of WT mice using
magnetic beads following the manufacturer's instructions (Miltenyi,
Auburn, Calif.). Splenocytes from OT-1 mice were activated with OVA
peptide for 3 days, washed with phosphate buffered saline (PBS),
and further cultured with mouse IL-2 (1 nM, Peprotech) for 2 days.
Expanded cells were more than 99% CD8 T cells. NK cells were
differentiated from the bone marrow of a WT mouse using 25 nM human
IL-15 (Peprotech). The culture consisted of >95% NK cells
(NK1.1.sup.+, CD3.sup.-) after depletion of NKT cells using
anti-CD3 magnetic beads (Miltenyi). EL4 murine lymphoma cell lines
were obtained from American Type Culture Collection (Manassas,
Va.). B16 murine melanoma cells expressing OVA were a gift from
Drs. John Frelinger and Edith Lord.
Murine granulocyte-colony stimulation factor (G-CSF) and
granulocyte macrophage-colony stimulation factor (GM-CSF), and
human interleukin 15 (IL-15) were purchased from Peprotech (Rocky
Hill, N.J.). Murine stem cell factor (SCF), Fms-related tyrosine
kinase 3 ligand (FLT3L) and thrombopoietin (TPO) were purchased
from R&D Systems (Minneapolis, Minn.). BM cells were flushed
from femurs and tibias of mice. The cells were cultured in RPMI
1640 media (Life Technologies, Grand Island, N.Y.), supplemented
with 2 mM L-glutamine, 100 IU/mL penicillin, 100 .mu.g/mL
streptomycin (Life Technologies), 10% fetal calf serum (Gemini Bio
Products, Sacramento, Calif.) and 50 .mu.M 2-mercaptoethanol (Sigma
Chemical, St. Louis, Mo.), at 37.degree. C. in 5% CO.sub.2.
Antibodies and Reagents
Antibodies were purchased from eBiosciences (San Diego, Calif.).
LPS was purchased from Sigma Aldrich. Naive OT-1 CD8 T cells were
labeled with 5-chloromethylfluorescein diacetate (CMFDA, Life
Technologies) following the manufacturer's instruction before
transfer to WT mice.
Activation of .sup.89Zr-oxine Complex Labeled Cells
After .sup.89Zr-oxine complex labeling, DCs were stimulated with
LPS (5 ng/ml) overnight. CD8 T cells labeled with .sup.89Zr-oxine
complex were stimulated with plate coated anti-CD3 antibody (10
.mu.g/ml) and anti-CD28 antibody (5 .mu.g/ml) for 3 days, then
transferred to an antibody-free culture supplemented with human
IL-15 (3 nM).
Determination of Viability of Cells and Release of .sup.89Zr from
Dead Cells
One million DCs or CTLs were labeled with .sup.89Zr-oxine complex
and washed. Cells were cultured in medium supplemented with GM-CSF
(20 .mu.g/ml) for DCs or TCR-activated for CTLs. At various time
points, the number of surviving cells was counted by a Countess
Automated Cell Counter (Invitrogen Corp., Carlsbad, Calif.) using
the trypan blue exclusion method. Using another set of labeled
cells, radioactivities of the cell pellet were measured by a
.gamma.-counter to determine the activity released from the cells
vs. activity retained in the cells. Three activity standards were
also counted each time for the decay correction.
Determination of the Functionality of .sup.89Zr-oxine Complex
Labeled Cells
CTLs, both labeled and unlabeled, were activated by plate coated
anti-CD3 antibody (10 .mu.g/ml) and anti-CD28 antibody (5 .mu.g/ml)
for 1.5 day and expression of CD3, CD8, CD44, CD25, CD69,
INF-.gamma. and IL-2 which was evaluated by flow cytometry. Bone
marrow-derived DCs on day 6 of culture with or without
.sup.89Zr-oxine complex labeling were stimulated with LPS (1 ng/ml)
overnight and surface expression of CD11c, CD80, CD86, CD40, MHC
class I and MHC class II was examined. In another experiment,
.sup.89Zr-labeled DCs were stimulated with LPS in the presence of
OVA overnight, transferred to Rag1KO mice expressing Ly5.2 (2
million cells) pre-injected with CMFDA labeled OT-1 CD8 T cells
expressing Ly5.1. Peripheral blood and splenocytes were collected
3.5 days later and dilution of CMFDA in OT-1 T cells was analyzed
using flow cytometry gated on Ly5.1+CD8 T cells.
Tracking of the .sup.89Zr-Labeled DCs and T Cells by MicroPET
Five million .sup.89Zr-labeled DCs and CTLs (148-185 kBq or 4-5
.mu.Ci in 200 .mu.l PBS) were transferred to mice via a tail vein
injection. Imaging was performed using a microPET/CT imager
(BioPET, Bioscan, Washington, D.C.) up to 7 days after the
injection. Using a 400-700 keV energy window, a 5-90 min-emission
scan per bed position; a total of two bed positions were scanned at
0 h-day 7. Images were reconstructed by a 3-dimensional
ordered-subsets expectation maximization (3D-OSEM) algorithm. The
maximum intensity projection images were fused with CT images using
InVivoScope software (Bioscan, Washington, D.C.).
Statistical Analysis
All experiments were performed in triplicates or repeated more than
three times.
The P-values were calculated by Friedman test for analysis of
.sup.89Zr-oxine labeled BM cell survival. Wilcoxon matched-pairs
signed rank test was used to analyze .sup.89Zr total activity
retention and specific activity in the cells. Two-way analysis of
variance followed by Tukey's multiple comparisons test correction
was used for examining the effect of plerixafor and G-CSF on BM
homing, and an unpaired two-tailed t-test for analyzing the effects
of plerixafor/G-CSF BM mobilization into the blood.
The P-values were calculated by two-way ANOVA using GraphPad Prism
software (GraphPad Software, Inc., La Jolla, Calif.) and P-values
less than 0.05 were considered significant.
EXAMPLE 1
This example demonstrates a synthesis of .sup.89Zr-oxine complex,
in accordance with an embodiment of the invention.
.sup.89Zr was produced at the institutional Cyclotron Facility
utilizing the nuclear reaction Y(p, 2n).sup.89Zr and an in-house GE
PETtrace beam-line (GE Healthcare) (L. Szajek et al., J. Nucl. Med.
Meeting Abstracts, 2013, 54: 1015). Briefly, adapting a previously
described method (J. P. Holland et al., Nucl. Med. Biol., 2009, 36:
729-739), yttrium metal mesh (200 mg) target cups were bombarded
with 13 MeV protons on a GE PETtrace. The irradiated target metal
was dissolved with 6N hydrochloric acid (HCl, 2 ml), and 10 M
hydrogen peroxide (0.1 ml) at 100.degree. C. for 1 h. After
dilution with water the .sup.89Zr solution was absorbed onto a
hydroxamate resin column. After washing the column with 2N HCl
followed by water, .sup.89Zr was eluted as oxalate with 1M oxalic
acid in greater than 96% radiochemical yield (<0.2% .sup.88Zr at
end of bombardment). .sup.89Zr-oxalate solution was loaded onto a
pre-treated C-18 Sep-Pak cartridge and washed with H.sub.2O.
.sup.89ZrCl.sub.4 was obtained after elution with 1N HCl (0.5
ml).
Non-radioactive zirconium-oxine standard was synthesized according
to a method reported previously using Zr (i-PrO).sub.4 in THF (P.
Kathirgamanathan et al., J Mater Chem, 2011, 21: 1762-1771). HPLC
was performed using a Beckman Gold HPLC system equipped with a
Model 126 programmable solvent module, a Model 168 variable
wavelength detector, a .beta.-Ram Model 4 radioisotope detector,
and Beckman System Gold remote interface module SS420X, using 32
KARAT.TM. software (Beckman Coulter, Brea Calif.). Analyses were
performed on a Waters STYRAGEL.TM. HT 1 (7.8.times.250 mm, 5 .mu.m)
column. Tetrahydrofuran (THF) solvent was used at 0.8 mL/min flow
rate. t.sub.R (Oxine)=9.34 min; t.sub.R (Zr-oxine)=8.0 min.
.sup.89Zr-oxine complex was generated by conjugating oxine to
.sup.89Zr. Oxine in 0.04N HCl (102 .mu.l, 20 mM) and
.sup.89ZrCl.sub.4 (60 .mu.l, 25.9-40.5 MBq or 700-1500 were mixed
in the presence of 4 .mu.l of 20% TWEEN.TM. 80. To this solution,
500 mM NaHCO.sub.3 (220 .mu.l) was added while vortexing to adjust
the pH to 7-7.2 and thereby allow chelation of .sup.89Zr by oxine
to take place while neutral oxine was released from its acidic
forms. To determine the conjugation yield, a small fraction of
reaction mixture was extracted with chloroform. The synthesis of
.sup.89Zr-oxine complex was accomplished with >97% yield, by
HPLC analysis and by determining radioactivity levels in the
chloroform and aqueous phases using a dose calibrator/gamma
counter.
EXAMPLE 2
This example demonstrates .sup.89Zr-oxine complex cell labeling in
accordance with an embodiment of the invention.
The cell labeling efficiency of .sup.89Zr-oxine complex was
examined in vitro as follows. A solution of .sup.89Zr-oxine complex
(88-740 kBq or 2.4-20 .mu.Ci) and cell suspension (1 million cells)
in PBS were incubated at room temperature (RT) for 15 min at 1:25
or 1:50 volume ratios. In some experiments, the labeling was
performed in serum free medium or in complete medium at 37.degree.
C. or at 4.degree. C. After the incubation, the cells were washed
with complete medium twice and transferred to a fresh tube and
washed again with PBS. Optionally, the labeled cells were washed,
separately or together, with solutions containing dilute
concentrations of scavenging chelating agents such as EDTA, DTPA,
or desferoxamine in order to remove any membrane-bound or loosely
bound .sup.89Zr resulting from incompletely internalized
.sup.89Zr-oxine complex.
For in vivo imaging, 5 million cells were labeled using 2.96 MBq of
.sup.89Zr-oxine complex per mouse at 1:25 volume ratios.
EXAMPLE 3
This example demonstrates that .sup.89Zr-oxine complex labeling
does not depend on active cellular incorporation.
To determine the optimal temperature for labeling cells with
.sup.89Zr-oxine complex, cell labeling was compared at 37.degree.
C., room temperature (RT) and 4.degree. C. using EL4 cells. One
million EL4 cells were incubated with .sup.89Zr-oxine complex at
1:50 volume ratios in PBS, serum free medium, or in complete medium
at 37.degree. C., room temperature, or 4.degree. C. for 15 min.
Radioactivities associated with the cells were determined, and the
results illustrated in FIG. 1A. The highest labeling efficiency was
achieved when cells were incubated with .sup.89Zr-oxine complex at
RT or at 4.degree. C. in PBS. This suggests that cell labeling does
not depend on cellular active transport. The use of serum free
medium did not significantly decrease the labeling compared to PBS
when labeling was performed at RT or at 4.degree. C. Using complete
cell media at RT or at 4.degree. C., the labeling efficiency
decreased to about two thirds of that achieved in PBS. The labeling
at 37.degree. C. was low under all media conditions.
EXAMPLE 4
This example demonstrates that .sup.89Zr-oxine complex-labeled DCs
and CTLs as examples of cells commonly used in cell-based
therapies.
Both cell types could be labeled with .sup.89Zr-oxine complex
although the labeling efficiency was higher with DCs (44%) than
CTLs, as illustrated in FIG. 1B. When naive and activated CTLs were
compared, naive T cells showed lower labeling efficiency (11%) than
activated CTLs (21%), which was also reflected in the specific
activity of the cells, as illustrated in FIG. 1C. NK cells grown
with IL-15 and EL4 mouse lymphoma cells showed higher labeling
efficiency and specific activity than primary CTL cells.
EXAMPLE 5
This example demonstrates that labeling with .sup.89Zr-oxine
complex did not interfere with cell survival or proliferation.
Because it is critical that the labeled cells remain viable and
functional, the survival of the cells after the labeling was
examined. DCs labeled with .sup.89Zr-oxine complex demonstrated
similar survival as compared to non-labeled control cells when
cultured with GM-CSF up to 5 days after the labeling (FIG. 2A). In
addition, proliferation was also examined for CTLs labeled with
.sup.89Zr-oxine complex. Labeled CTLs rapidly proliferated upon TCR
stimulation and underwent contraction when the stimulation was
terminated, similarly to the non-labeled controls (FIG. 2B).
Activity associated with .sup.89Zr-oxine complex bound to DCs was
stable and mirrored the number of DCs (FIGS. 2A and 2C). Total
radioactivity associated with the CD8 T cells did not decrease
while T cells underwent cell division (FIG. 2D), but decreased
during the contraction phase. These results suggest that the
.sup.89Zr, once incorporated, remains in the cells during the cell
division, but is likely released upon cell death.
EXAMPLE 6
This example demonstrates that labeling with .sup.89Zr-oxine
complex did not interfere with functionality of DCs and CTLs.
In order to determine the effect of .sup.89Zr-oxine complex on the
functionality of DCs and CTLs, labeled DCs were activated by LPS
and labeled CTLs were activated through TCR. .sup.89Zr-oxine
complex labeling resulted in slightly increased expression of B7
molecules, CD80 and CD86, on DCs prior to LPS activation. After
overnight stimulation with LPS, .sup.89Zr-oxine labeled DCs
upregulated CD80, CD86, and CD40, as well as MHC molecules (FIGS.
3A-E), similar to non-labeled control DCs. To confirm that the
labeled DCs were still capable of presenting antigens to T cells,
.sup.89Zr-oxine labeled DCs were stimulated with LPS together with
ovalbumin peptide (OVA), and injected into mice transferred with
CMFDA labeled OT-1 CD8 T cells expressing TCR against OVA 1 day
before DC administration. Flow cytometry analysis of OT-1 T cells
collected from the spleen and blood 4 days after the DC transfer
demonstrated that .sup.89Zr-oxine-labeled DCs (black line) were
capable of presenting the antigen and inducing T cell activation
and proliferation as well as non-labeled DCs (gray shadow) (FIG.
3F).
CTLs were TCR stimulated after the .sup.89Zr-oxine complex
labeling. TCR stimulation induced upregulation of CD69, CD25 and
CD44 markers in the labeled and non labeled CTLs at the similar
levels (FIGS. 4A-4C). The labeling did not negatively affect the
production of interferon gamma (IFN.gamma.) and Interleukin 2
(IL-2) (FIGS. 4D and 4E), suggesting that the cytotoxic functions
of CTLs were maintained after labeling.
EXAMPLE 7
This example demonstrates that .sup.89Zr-oxine complex labeling of
transferred DCs and CTLs enabled visualization on microPET.
DCs labeled with .sup.89Zr-oxine complex were visualized in vivo
with microPET imaging (FIG. 5A). Labeled DCs injected via the tail
vein (444 kBq or 12 .mu.Ci/5 million cells) initially distributed
in the lungs, and gradually migrated to the spleen and liver by day
1 (FIG. 5A). The DCs remained in the liver and spleen during the 7
day-imaging period. This distribution was confirmed by a
biodistribution study analyzing the radioactivity of each organ
harvested from the mice on day 1 and day 7 (FIG. 7). The low
activity shown in the kidneys and the bone (femur) was likely due
to free .sup.89Zr released from dead cells.
CTLs purified from the spleen of WT mice and labeled with
.sup.89Zr-oxine complex labeling (185 kBq or 5 .mu.Ci/5 million
cells) were tracked over 7 days. Unlike DCs, CTLs mainly
distributed in the spleen and lymph nodes after migrating out from
the lungs (FIG. 5B). Arrows indicate examples of lymph node
accumulation of CTLcytotoxic T cells.
EXAMPLE 8
This example demonstrates that .sup.89Zr-oxine complex-labeled CTLs
targeted tumor.
The tumor targeting properties of .sup.89Zr-oxine complex-labeled
CTLs were examined using ex vivo activated OT-1 CD8 T cells in a
B16 melanoma xenograft model. Rag1KO mice bearing B16 tumor
expressing OVA (B 16-OVA, .about.1 cm diameter) were injected with
one million splenocytes of WT mice 6 hours before the OT-1 T cell
transfer. The .sup.89Zr-oxine complex-labeled OT-1 T cells (248.5
kBq or 6.7 .mu.Ci/7.7 million cells) were adoptively transferred
and serial imaging was performed. FIG. 6A shows the migration of
OT-1 T cells to the tumor, which accumulated over time. The B16-OVA
tumor underwent regression after the .sup.89Zr-oxine
complex-labeled OT-1 T transfer, indicating that cytotoxic action
of CTLs was maintained after .sup.89Zr-oxine complex labeling,
leading to a dramatic response in the tumor (FIG. 6B). Untreated
mice were sacrificed on day 7, as the tumor diameter reached 20 mm
(n=5). *P=2.84.times.10-5 on day 7 according to Holm-Sidak multiple
tests that included three tests corresponding to days 2, 5, and 7
(FIG. 6B). Error bars indicate standard deviations.
EXAMPLE 9
This example demonstrates .sup.89Zr-oxine bone marrow (BM) cell
labeling and the determination of cell viability and cellular
retention of .sup.89Zr.
BM cells were incubated with 11.0-55.5 kBq/10.sup.6 cells of
.sup.89Zr-oxine complex in PBS at 25:1 volume ratios for 20
minutes, washed twice in RPMI media and transferred to a new tube.
The cell-associated radioactivity was 3.0-16.7 kBq, yielding the
labeling efficiency of 24-30%. To determine cell viability and
retention of .sup.89Zr after the labeling, BM cells were labeled
with 2 different radioactivity doses, 28.12 kBq/10.sup.6 and 8.14
kBq/10.sup.6 cells, and cultured with SCF, FLT3L and TPO (100 ng/ml
each). The number of live cells was counted using 0.4% trypan blue
dye (Life Technologies) at 0 h, 2 d, 4 d, and 7 d (n=3). At each
time point, the cell suspension was spun to separate the
supernatant and the cell pellet, and the radioactivity of both
fractions was measured by a .gamma.-counter (WIZARD.sup.2 automatic
gamma-counter, Perkin Elmer, Waltham, Mass.).
The culture with combination of SCF, FLT3L and TPO sustained the
survival of a fraction of BM cells as indicated in FIG. 8A.
.sup.89Zr-labeling at 8.14 kBq/10.sup.6 cells showed slight
proliferation after day 4, whereas cells labeled at 28.12
kBq/10.sup.6 cells slightly decreased in number. With the lower
labeling dose, the suppression of the proliferation was limited to
approximately 50% (global P=0.0417 against non-labeled control).
The total .sup.89Zr activities associated with the cells declined
as the cells not responding to the cytokines died during the
initial 0-2 day period, but plateaued thereafter (FIG. 8B; global
P=0.25). As the cells labeled with the lower dose started to
proliferate, the specific activity declined, but specific activity
for the cells labeled with the higher dose remained about the same
as they failed to proliferate (FIG. 8C; global P=0.25).
EXAMPLE 10
This example demonstrates the determination of the effects of
labeling on the phenotype of BM cells and differentiation
capability in vitro.
The surface expressions of CD117, sca-1 and lineage markers (CD3,
NK1.1, Ly6G, CD2, CD5 and B220) on BM cells were examined before
and after .sup.89Zr-oxine labeling (3.15 kBq/2.times.10.sup.6) and
analyzed by flow cytometry using a FACS Calibur flow cytometer
(Becton Dickinson, San Jose, Calif.) and analyzed using FlowJo
software (Tree Star, Inc., Ashland, Oreg.). BM cell differentiation
capability was examined by culturing the .sup.89Zr-labeled and
non-labeled cells with GM-CSF (20 ng/ml) and IL-15 (25 nM) for
differentiation to DCs and NK/NK-T cells (n=3). On day 10, the
cells were harvested, stained with anti-CD11c, CD86, and NK1.1
antibodies, and analyzed on flow cytometry. For the GM-CSF culture,
cell associated-.sup.89Zr activity and cell number were also
examined.
.sup.89Zr-oxine labeled cells expressed CD117, sca-1 and lineage
markers (CD3, NK1.1, Ly6G, CD2, CD5 and B220) at similar levels to
non-labeled controls (FIG. 9A). CD117.sup.+sca-1.sup.+ HSCs were
4.7-4.8% of lineage negative cells, and slightly more than 0.4% of
the total BM cells. GM-CSF culture of BM Cells, labeled at 39.3 and
26.9 kBq/10.sup.6 cells, demonstrated similar survival and minimal
suppression of proliferation in comparison to non-labeled controls
during the differentiation into DCs. Because most BM cells were
non-responders to GM-CSF and died, retention of .sup.89Zr in whole
culture rapidly decreased during the initial 3 day-period and
remained low thereafter. After a 10 day-culture with GM-CSF, the BM
cells labeled with .sup.89Zr-oxine differentiated to CD11c.sup.+
DCs with about half of the cells expressing CD86, indicating that
they are mature DCs (FIG. 9B). When IL-15 was used, cells became
NK1.1.sup.+ cells, suggesting their differentiation into
NK.sup./NK-T cells (FIG. 9C). These results indicated that
.sup.89Zr-oxine labeled cells retained the capacity to proliferate
and differentiate normally into mature cells in vitro.
EXAMPLE 11
This example demonstrates BM cell tracking by microPET/CT.
.sup.89Zr-oxine labeled BM cells were transferred to mice i.v. at
331 kBq/2.times.10.sup.7 cells. For BM ablation, host mice received
a 9.5 Gy lethal whole-body irradiation 24 h prior to cell transfer
(n=5). In addition, mice received deferoxamine, a chelator,
(Hospira, Inc., Lake Forest, Ill.) at 660 .mu.g intramuscularly 15
min before and 1, 2, 3 and 4 hr after the cell injection to hasten
the renal excretion of free .sup.89Zr released from cells that died
after the transfer. Imaging was performed using a microPET/CT
imager (BioPET, Bioscan, Washington, D.C.) up to 7 days (Suppl.
Methods). InVivo Quant software (inviCRO LLC, Boston, Mass.) was
used to fuse the maximum intensity projection PET images with CT
images and to quantify cells migrated to various organs by setting
volumes of interest on the acquired images.
It was observed that the donor cells quickly passed through the
lungs, with a small fraction of cells migrating to the BM, spleen
and liver almost immediately following the transfer (FIGS. 10A and
10B). The majority of donor cells rapidly left the lungs within 4 h
and migrated to the BM and spleen and remained in these organs
until day 7. BM ablation prior to the cell transfer was performed
in some animals (n=3). Subsequent PET/CT imaging showed no clear
difference in the initial migration of the donor cells to the BM
comparing BM ablated and non-ablated mice and trafficking pattern
of the two groups remained similar up to day 7 (FIG. 10A). The
analysis of cell migration kinetics to the BM, spleen and liver in
both groups were also similar (n=3, FIG. 10B). It was calculated
that when 2.times.10.sup.7 cells were transferred, approximately
4.4.times.10.sup.6 cells homed to the BM and 2.3.times.10.sup.6 and
7.1.times.10.sup.6 cells to the spleen and liver, respectively, by
4 h (FIG. 10B).
EXAMPLE 12
This example demonstrates role of CXCR4 in the BM cell
trafficking.
A CXCR4 inhibitor, plerixafor (Adooq Bioscience, Irvine, Calif.),
was used to interrogate the role of CXCR4 in BM cell migration
(n=4). Mice received an i.v. injection of plerixafor (5 mg/kg) 15
min before and 1 day after the .sup.89Zr-oxine labeled BM cell
transfer (331 kBq/2.times.10.sup.7 cells). Another group of mice
received concurrent i.v. injections of G-CSF (2.5 .mu.g, n=4).
Deferoxamine was injected intramuscularly 15 min before and 1, 2, 3
and 4 hr after the cell injection. Serial microPET/CT images were
acquired.
Inhibition of CXCR4 by plerixafor significantly inhibited the
migration of labeled cells to the BM at 2 h (FIGS. 11A and 11B)
indicating that CXCR4 signaling is critical for BM homing. This
blockade of the initial BM homing lasted even longer when G-CSF was
additionally administered (FIGS. 11A and B). Based on the
trafficking kinetics analyzed from the PET images, approximately
4.9.times.10.sup.5 cells were mobilized by plerixafor injected on
day 1, suggesting that their retention in the BM also depended on
CXCR4-CXCL12 system (FIG. 11B). Similarly, the combination of
plerixafor/G-CSF mobilized approximately 1.04.times.10.sup.5 cells
on day 1, which indicated that even the donor cells homed to the BM
by day 1 under the plerixafor/G-CSF treatment was low, yet further
treatment still forced the BM cells to mobilize out of the BM.
EXAMPLE 13
This example demonstrates the quantification of BM cell
mobilization.
BM cells collected from GFP transgenic mice (222
kBq/2.times.10.sup.7 cells) were labeled with .sup.89Zr-oxine and
transferred to wild type mice (n=4). Mice received i.v. injections
of plerixafor and G-CSF 3 h and 1 day following the cell transfer.
Two hours after the second mobilization treatment, mice were
sacrificed by CO.sub.2 asphyxiation and the blood was collected by
a cardiac puncture into EDTA-coated tubes (BIOTANG Inc., Lexington,
Mass.). The volume was measured, then the blood was spun and the
radioactivity of the cell fraction was measured by a
.gamma.-counter. Radioactivity of the total blood was calculated
using the following formula using the 1.1 ml average blood volume
for a 20-g mice; (Circulating .sup.89Zr-labeled BM cells)=(Total
injected cell number).times.(radioactivity of the blood
sample)/(Total injected radioactivity).times.1.1 (ml)/(sample blood
volume [ml]).times.body weight (g)/20. The collected blood cells
were also analyzed by flow cytometry.
The strong effect of plerixafor and G-CSF in inhibiting BM homing
and further inducing mobilization of BM cells prompted
quantification of the mobilization effect of plerixafor/G-CSF on
pre-transplanted BM cells. The injection of plerixafor/G-CSF on two
consecutive days induced a 3.5-fold increase of .sup.89Zr activity
in the circulation in mice pre-transplanted with .sup.89Zr-labeled
BM cells (FIG. 12A; P=0.0192), corresponding to an increase from
0.31.times.10.sup.5 cells to 1.1.times.10.sup.5 cells in the
circulation by the mobilization. The cell number calculated from
the blood radioactivity was consistent with what was estimated from
the PET image-quantification above. Because the .sup.89Zr-labeled
cells in the spine were about 4.4.times.10.sup.6, approximately 2%
of the BM cells were mobilized by the 2 doses of plerixafor/G-CSF
treatment. Flow cytometry analyses confirmed an increase of
GFP.sup.+ cells in the blood in mice treated with plerixafor/G-CSF
compared to controls (FIG. 12B).
EXAMPLE 14
This example demonstrates the flow cytometry analysis for
engraftment of .sup.89Zr-oxine labeled BM cells and differentiation
in vivo.
The engraftment and differentiation of the .sup.89Zr-oxine labeled
BM cells was examined by transferring CD45.1 expressing donor cells
to CD45.2 expressing hosts or vice versa (n=3). Ten-weeks later,
the BM cells and splenocytes collected from the recipient mice were
stained with antibodies against CD45.1 and CD45.2 congenic markers.
Splenocytes were also stained with antibodies against CD3, CD4,
CD8, NK1.1, and CD11c. Cells were analyzed by flow cytometry.
Flow cytometry analysis of recipient BM revealed that
.sup.89Zr-labeled donor cells had engrafted only when the host mice
had received whole-body irradiation before the BM transfer (FIGS.
13A and 13B) In the BM ablated mice, 79% of BM cells were
.sup.89Zr-labeled donor cell origin (FIG. 13Ai) and phenotypically
similar to the control mice that received unlabeled BM cells. In
the periphery, about 90% of splenocytes consisted of donor-derived
cells (FIG. 13Bi), which included mature DCs (CD11c.sup.+, FIG.
13Bii), and T cells (CD3.sup.+NK1.1.sup.-, FIG. 13Biii), and NK
cells (CD3.sup.-NK1.1.sup.+, FIG. 13Biv). These results suggest
that critical functions of BM cells, such as homing capacity to the
BM, engraftment in the BM niche, and differentiation into mature
cells, were retained in .sup.89Zr-oxine labeled cells.
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and "at least one" and
similar referents in the context of describing the invention
(especially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments can become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *